Supplemental Table : Strains used in this study Name Parent Genotype Reference GA-8 MATa, ade-; can-; his3-,5; trp-; ura3-; leu-3, W33-A GA-3 GA-8 his3-,5::gfp-laci-his3, NUP49::GFP-NUP49-URA3 [] GA-46 GA-3 + ARS67::ARS67 - laco-trp [] GA-95 GA-46 esc::kanmx This study GA-84 GA-46 ptlc-δ48int [as in 3] This study GA-369 GA-3 ARS54::ARS54 - laco-lexa-trp [4] GA-98 MATa, ade-; can-; his3-,5; trp-; ura3-; leu-3, W33-A GA-49 GA-98 nup33::his3 This study GA-438 GA-49 (pun-nup33δn - LEU) This study GA-458 GA-438 pun-nup33δn - leu::kanmx (marker swap) This study GA-45 GA-458 NUP49::CFP-NUP49 - URA3 This study GA-4583 GA-45 +ade-::laci-gfp::ade This study GA-4584 GA-4583 lys::laco.lexa-trp This study GA-46 W33-A +telomere 5R::ADE This study GA-37 GA-46 +tel::his5 This study GA-9 GA-46 +yku7::kanmx This study GA-575 GA-46 MPS3::mps3Δ75-5-KanMx6 [as in 5] This study GA-4954 GA-369 +sir4::hphmx This study GA-3467 GA-5559 W33-A/α GA-46 GA-3467 MATa/MATα, ade-; can-; his3-,5; trp-; ura3-; leu-3, EST/est::HphMX Diploid; see parental phenotypes This study This study Strain references. Heun, P., Laroche, T., Raghuraman, M.K., and Gasser, S.M. (). The positioning and dynamics of origins of replication in the budding yeast nucleus. J Cell Biol 5, 385-4.. Hediger, F., Neumann, F.R., Van Houwe, G., Dubrana, K., and Gasser, S.M. (). Live imaging of telomeres: yku and Sir proteins define redundant telomere-anchoring pathways in yeast. Curr Biol, 76-89. 3. Stellwagen, A.E., Haimberger, Z.W., Veatch, J.R., and Gottschling, D.E. (3). Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev 7, 384-395. 4. Schober, H., Kalck, V., Vega-Palas, M.A., Van Houwe, G., Sage, D., Unser, M., Gartenberg, M.R., and Gasser, S.M. (7). Controlled exchange of chromosomal arms reveals principles driving telomere interactions in yeast. Genome Res.
5. Bupp, J.M., Martin, A.E., Stensrud, E.S., and Jaspersen, S.L. (7). Telomere anchoring at the nuclear periphery requires the budding yeast Sad-UNC-84 domain protein Mps3. J Cell Biol. Supplemental Material Simulation and computational modeling The theoretically expected random colocalization of a spot with the pore cluster was calculated geometrically. If the center of the spot is in a certain region of the nucleus the spot is considered as colocalizing with the cluster. If the spot is distributed uniformly inside the nucleus then the is the ratio C/V where C is the volume of the region mentioned above and V is the total volume that is available to the spot. The pore cluster was modeled as a conical layer inside the nucleus (see Supplemental Figure ) and the spot was considered as colocalizing if it at least touches the cluster. The total available volume is the volume of the nucleus minus the volume of the nucleolus (estimated as 3%). Furthermore, the is affected by the position of the spot relative to the nuclear periphery. We calculated this effect by dividing the nucleus into the outermost shell (corresponding to zone ) and the interior (zone and 3). If of N spots εn are in zone then the ς can be calculated as ς = ες + ( - ε)ς 3 where ς = C /V and ς 3 = C 3 /V 3 are the volume fractions for colocalization in zone and zone and 3, respectively. See Supplemental Figure for details on the calculation of ς. Supplemental Figure (A) The pore cluster is modeled as a conical disk at the periphery of the nucleus. The thickness d, the diameter l, and the radius of the spot r where measured in 3D reconstructions of microscopic images. A possible outline of the nucleus is shown for illustration. (B) The for a random spot was calculated analytically as a fraction of volumes (see Supplemental methods). The figure shows a cut through the model. The pore cluster is a conical layer shown in red. The spot is considered as colocalizing if it at least touches the pore cluster which results in the colocalization region shown in green. For the calculation this region is approximated again by a conical
layer outlined in dark green (see blow up in the left part of the figure). In the parameter range used here this causes an error in the volume calculation of less than 5%. The nucleolus is shown in grey and is calculated as roughly 3% of the nuclear volume. It is assumed not to overlap with the pore cluster and under this condition its exact position is irrelevant to the calculation. Zone (see text) is shown in light blue. We use the following identifiers, all relative lengths are normalized to R, all relative volumes are normalized to V tot = 4/3πR 3 : R: nuclear radius d: thickness of the colocalization volume ρ d : (R - d) / R ρ = (/3) /3 : relative inner radius of zone θ: angle determining the size of the colocalization volume η: relative height of the nucleolus κ = /4 η (3 - η): relative volume of the nucleolus ε: fraction of spots in zone Note that d is the thickness of the colocalization volume which is not identical to the thickness d of the cluster: d = d + r where r is the radius of the spot. Likewise we have to calculate θ from l and r: θ = θ + sin - (r/r) where θ = sin - (l /R). Without enrichment in zone (i.e. ε = /3) the ς can be calculated as ς = ( ρ d )( cosθ ). κ With enrichment in zone this has to be replaced by ς = ε 3 κ + 4 3 ( ρ )( cosθ ) ( η + ρ ) ( ρ η + ) + ( ε ) 3 ρ 4 3 3 ( ρ ρ d )( cosθ ) ( η + ρ ) ( ρ η + ) Two case differentiations are necessary during the derivation of the above formula. In the given form it assumes that both the nucleolus and the colocalization volume extend into zone, i.e. η > ρ and ρ > ρ d, respectively. (C) Expected colocalization versus cluster thickness for three different cluster diameters. The curves do not start at a colocalization value of zero because even for a very thin cluster the colocalization volume is finite. When the cluster thickness approaches the nuclear radius the colocalization volume increases very slowly leading to a very flat.
curve. The dashed lines show the measured cluster thickness and the smallest measured colocalization. (D) Expected colocalization versus cluster diameter for three different cluster thicknesses. The vertical dashed line shows the measured cluster diameter. (E) Expected colocalization versus volume fraction occupied by the nucleolus. The increases when the total accessible volume decreases. This can be the case if a certain volume fraction is occupied e.g. by the nucleolus. The vertical dashed line indicates a volume fraction of one third. Cluster thickness and diameter have their default values of 34nm and 8nm, respectively. (F) Expected colocalization versus enrichment in zone. If the spot is more likely to be at the periphery this also increases the expected unspecific colocalization. The vertical dashed lines indicate the uniform distribution (fraction /3) and a typical enriched value of.6. Supplemental Figure : Mlp and Mlp are not required for Yku8 anchoring Targeted anchoring of internally localized ARS67 bearing laco and lexa binding sites was scored in strains GA-765 (W33-A, mlpδ mlpδ, GFP-NUP49, laci-gfp, lacolexa sites). GA-765 + lexa alone ((G+S) n=), GA-765 + lexa-yku8 ((G+S) n=93), and GA-765 + lexa-yku8-4 ((G+S) n=3). * indicates significantly different than random. G and S phase results were not significantly different and were therefore combined. Supplemental Figure 3: Tel6R anchoring is lost in sir4 and est mutants The position of Tel6R tagged with laco sites was scored relative to three zones as described in Figure, in the following strains: GA-459 (Tel6R ) (G n= 87, S phase n=6); GA-867 (Tel6R sir4δ) (G phase n=6, S phase n=57) and GA-867-estΔ (Tel6R estδsir4δ) (G n=5, S phase n=36). * indicates significantly different than random and P values are given. Supplemental Figure 4: tel cells lose sliencing efficiency Telomeric position effect or silencing was monitored in cells bearing the ADE marker at Tel5R in vs telδ cells. GA-46 (W33, ) is restreaked next to isogenic telδ strain GA-37. The loss of pink color indicates loss of repression.
A B Average width d = 34nm, n=5 ρ d R pore cluster θ spot Average length l = 8nm, n=63 Spot diameter r = 5nm, n=9 ρ R zone nucleolus ηr C D.3.3.5..5. cluster diameter 6nm cluster diameter 8nm cluster diameter nm.5..5. cluster thickness nm cluster thickness 34nm cluster thickness 48nm.5.5 4 6 8 cluster thickness [nm] 4 6 8 4 cluster diameter [nm] E F.3.3.5.5..5...5..5.5...3.4.5..4.6.8 volume fraction occupied by the nucleolus fraction of spots in zone Schober et al Supplemetal Figure
ARS67 lexa in mlp/δ lexa-yku8 in mlp/δ lexa-yku8-4 in mlp/δ random * random % foci 8 6 4 Zone G and S phase * * 3 Schober et al. Supplemental Figure Tel6R 8 P =.48 G phase 8 P =. S phase laci-gfp sir4δ sir4δ estδ % foci 6 4 Zone 3 6 4 P =.76 * 3 Schober et al. Supplemental Figure 3
Sir mediated repression telomere 5R ADE TG (-3) 3-35bp tel telomere 5R ADE TG (-3) -5 bp Schober et al Supplemental Figure 4